Compounds for fluorescence labeling

ABSTRACT

There are provided fluorescent compounds that can be used for DNA sequencing, measurement of physiologically active substance or the like based on fluorescence immunoassay and so forth. 
     Compounds represented by the formula (I) (V 1  to V 6  represent a hydrogen atom or a functional group selected from the group consisting of a halogen atom, an alkyl group, an alkenyl group, a group that can form a covalent bond with a compound to be labeled and the like, and V 1 , V 2  and others may bind to each other to form a saturated or unsaturated ring; R 1  represents a hydrogen atom or a functional group selected from the group consisting of an alkyl group, an aryl group and a heterocyclic group; R 3  and R 4  represent an alkyl group, and R 3  and R 4  may bind to each other to form a ring; Q represents a group of atoms required to form a methine dye chromophore; and m and n represent 0 or 1, provided that m+n is 1) or salts thereof

This application is a 371 of PCT/JP00/0640, filed Sep. 20, 2000.

TECHNICAL FIELD

The present invention relates to highly sensitive fluorescence-labelingagents, which are used for DNA sequencing or measurement ofphysiologically active substance or the like based on fluorescenceimmunoassay, or used as a fluorescence contrast medium that isadministered into a blood vessel to visualize information in vivo andthe like. The invention also relates to synthetic intermediates for saidagents.

BACKGROUND ART

For DNA sequencing and measurement of physiologically active substanceor the like based on fluorescence immunoassay, methods haveconventionally been used which comprise the step of labeling a targetsubstance with a radioisotope. However, these methods have problems fromviewpoints of safety, storability of agents or the like, and therefore,various methods for labeling a target substance with a fluorescent dyehave been studied as alternatives to the above methods. Performancesrequired for fluorescence-labeling agents include, for example, (1)agents should have a high fluorescence quantum yield, (2) should have ahigh molecular extinction coefficient, (3) should be water-soluble andshould not aggregate in an aqueous solvent to cause self-quenching, (4)should be hardly hydrolyzed, (5) should hardly cause photodegradation,(6) should be hardly influenced by background fluorescence, and (7)should be introduced with a reactive substituent that produces acovalent bond with a target substance.

Fluorescein isothiocyanate (FITC) and rhodamine isothiocyanate, whichhave been known as fluorescence-labeling agents since old days, have ahigh fluorescence quantum yield. However, they have a low molecularextinction coefficient. In addition, they suffer from a problem thatthey are susceptible to background fluorescence of a membrane used forblotting since their excitation and emission wavelengths are within therange of 500 nm to 600 nm.

As dyes having a high molecular extinction coefficient, for example,cyanine dyes disclosed in U.S. Pat. No. 5,486,616, Japanese PatentUnexamined Publication (Kokai) Nos. 2-191674, 5-287209, 5-287266,8-47400, 9-127115, 7-145148 and 6-222059, and polymethine dyes such asoxonol barbiturate disclosed in Journal of Fluorescence, 5, p.231 (1995)have been known. However, these dyes generally have a problem of a lowfluorescence quantum yield. Further, these dyes also have a problem thatthey are hardly soluble in water and that they are hydrolyzed even ifthey are dissolved in water. In addition, they also suffer from aproblem that they form aggregates in an aqueous medium due to theirstrong intermolecular interaction among dye molecules and thusself-quenching of fluorescence is often observed.

The cyanine dyes disclosed in Japanese Patent Unexamined Publication No.2-191674 and other publications are superior dyes with water-solubilityand suppressed formation of aggregates based on introduction ofsulfonate group into a relatively stable chromophore. However, the dyesdo not have satisfactorily high fluorescence quantum yield, and theysuffers from a problem that synthesis of the dyes becomes difficult dueto the introduction of sulfonate group. Under the circumstances,development of fluorescent dyes has been desired which have highwater-solubility and stability and are free from self-quenching offluorescence due to aggregation, as well as characteristic of strongfluorescence.

As other dye backbone structure with strong fluorescence, azaindoleninecyanine dyes disclosed in British Patent No. 870,753 have been known.However, the patent publication is silent about characteristicsessential for fluorescence-labeling agents such as water-solubility,aggregation property and stability of aqueous solutions. Furthermore, noexample is given as to introduction of a reactive substituent thatproduces a covalent bond with a target substance. Therefore, theirsuitability as fluorescence-labeling agents remains completely unknown.Further, Japanese Patent Unexamined Publication Nos. 4-358143, 3-195668,1-280750 and European Patent Laid-open EP841958 disclose applications ofthe azaindolenine cyanines for photographic purpose. However, theaforementioned applications utilize absorption characteristics of theazaindolenine cyanines, and do not consider and actively utilize theirluminescence characteristics.

DISCLOSURE OF THE INVENTION

An object of the present invention is to provide a compound that isuseful as a highly sensitive fluorescence-labeling agent used for DNAsequencing or measurement of physiologically active substances or thelike based on fluorescence immunoassay, or used as a fluorescencecontrast medium that is administered into a blood vessel to visualizeinformation in vivo. Another object of the present invention is toprovide a compound useful as synthetic intermediates for preparation ofthe aforementioned compounds. The inventors of the present inventionconducted various studies to achieve the aforementioned objects, and asa result, they successfully achieved the aforementioned objects byproviding the following compounds.

The present invention thus provides compounds represented by thefollowing general formula (I) or salts thereof:

wherein, V¹, V², V³, V⁴ and V⁵ each independently represent a hydrogenatom or a group selected from the group consisting of a halogen atom, analkyl group, an alkenyl group, an alkynyl group, an aryl group, aheterocyclic group, cyano group, a hydroxy group, nitro group, carboxylgroup, an alkoxy group, an aryloxy group, a silyloxy group, aheterocyclyloxy group, an acyloxy group, a carbamoyloxy group, analkoxycarbonyloxy group, an aryloxycarbonyloxy group, an amino group(including an anilino group), an acylamino group, an aminocarbonylaminogroup, an alkoxycarbonylamino group, an aryloxycarbonylamino group, asulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylaminogroup, a mercapto group, an alkylthio group, an arylthio group, aheterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, anarylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, anacyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, acarbamoyl group, a phosphono group, a phosphonato group and a group thatcan form a covalent bond with a compound to be labeled (each of saidgroup may be substituted), provided that V¹, V², V³, V⁴ and V⁵ do notsimultaneously represent a hydrogen atom and provided that V¹ and V², V²and V³, and V⁴ and V⁵ may independently bind to each other to form asaturated or unsaturated ring that may be substituted; R¹ represents ahydrogen atom or a group selected from the group consisting of an alkylgroup, an aryl group and a heterocyclic group (each of said group may besubstituted); R³ and R⁴ represent an alkyl group that may besubstituted, and R³ and R⁴ may bind to each other to form a ring thatmay be substituted; Q represents a group of atoms required to form acyanine dye chromophore, a melocyanine dye chromophore or a stilyl dyechromophore; and m and n represent 0 or 1, provided that m+n is 1.

The present invention also provides compounds represented by thefollowing general formula (II):

wherein, V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, V⁹ and V¹⁰ each independentlyrepresent a hydrogen atom or a group selected from the group consistingof a halogen atom, an alkyl group, an alkenyl group, an alkynyl group,an aryl group, a heterocyclic group, cyano group, hydroxy group, nitrogroup, carboxyl group, an alkoxy group, an aryloxy group, a silyloxygroup, a heterocyclyloxy group, an acyloxy group, a carbamoyloxy group,an alkoxycarbonyloxy group, an aryloxycarbonyloxy group, an amino group(including an anilino group), an acylamino group, an aminocarbonylaminogroup, an alkoxycarbonylamino group, an aryloxycarbonylamino group, asulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylaminogroup, a mercapto group, an alkylthio group, an arylthio group, aheterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, anarylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, anacyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, acarbamoyl group, a phosphono group, phosphonato group and a group thatcan form a covalent bond with a compound to be labeled (each of saidgroup may be substituted), provided that V¹, V², V³, V⁴ and V⁵ do notsimultaneously represent a hydrogen atom, and provided that V¹ and V²,V³ and V³, V⁴ and V⁵, V⁶ and V⁷, V⁷ and V⁸, and V⁹ and V¹⁰ may eachindependently form a saturated or unsaturated ring; R¹ and R² eachindependently represent a hydrogen atom or a group selected from thegroup consisting of an alkyl group, an aryl group and a heterocyclicgroup (each of said group may be substituted); R³, R⁴, R⁵ and R⁶ eachindependently represent an alkyl group that may be substituted, and R³and R⁴, and R⁵ and R⁶ may bind to each other to independently form aring that may be substituted; m, n, s and t represent 0 or 1, providedthat m+n is 1 and s+t is 1; L¹, L² and L³ each independently represent amethine group that may be substituted; p represents 1, 2 or 3; Mrepresents a counter ion, and q represents a number required toneutralize the charge of the molecule.

The present invention further provides compounds represented by thefollowing general formula (III) or salts thereof:

wherein, V¹, V² and V³ each independently represents a hydrogen atom ora group selected from the group consisting of a halogen atom, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, a heterocyclicgroup, cyano group, hydroxy group, nitro group, carboxyl group, analkoxy group, an aryloxy group, a silyloxy group, a heterocyclyloxygroup, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxygroup, an aryloxycarbonyloxy group, an amino group (including an anilinogroup), an acylamino group, an aminocarbonylamino group, analkoxycarbonylamino group, an aryloxycarbonylamino group, asulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylaminogroup, a mercapto group, an alkylthio group, an arylthio group, aheterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, anarylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, anacyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, acarbamoyl group, a phosphono group, a phosphonato group and a group thatcan form a covalent bond with a labeling compound (each of said groupmay be substituted), provided that V¹, V² and V³ do not simultaneouslyrepresent a hydrogen atom, and provided that V¹ and V², and V² and V³may each independently form a saturated or unsaturated ring that may besubstituted; R¹ represents a hydrogen atom or a group selected from thegroup consisting of an alkyl group, an aryl group and a heterocyclicgroup (each of said group may be substituted); R³ and R⁴ eachindependently represent an alkyl group that may be substituted, and R³and R⁴ may bind to each other to form a ring that may be substituted;and compounds represented by the following general formula (IV):

wherein V¹, V², V³, V⁶, V⁷ and V⁸ represent a hydrogen atom or a groupselected from the group consisting of a halogen atom, an alkyl group, analkenyl group, an alkynyl group, an aryl group, a heterocyclic group,cyano group, hydroxy group, nitro group, carboxyl group, an alkoxygroup, an aryloxy group, a silyloxy group, a heterocyclyloxy group, anacyloxy group, a carbamoyloxy group, an alkoxycarbonyloxy group, anaryloxycarbonyloxy group, an amino group (including an anilino group),an acylamino group, an aminocarbonylamino group, an alkoxycarbonylaminogroup, an aryloxycarbonylamino group, a sulfamoylamino group, analkylsulfonylamino group, an arylsulfonylamino group, a mercapto group,an alkylthio group, an arylthio group, a heterocyclylthio group, asulfamoyl group, an alkylsulfinyl group, an arylsulfinyl group, analkylsulfonyl group, an arylsulfonyl group, an acyl group, anaryloxycarbonyl group, an alkoxycarbonyl group, a carbamoyl group, aphosphono group, a phosphonato group and a group that can form acovalent bond with a compound to be labeled (each of said group may besubstituted), provided that V¹, V² and V³ do not simultaneouslyrepresent a hydrogen atom, and provided that V¹ and V², V² and V³, V⁶and V⁷, and V⁷ and V⁸ may each independently form a saturated orunsaturated ring that may be substituted; R¹ and R² each independentlyrepresent a hydrogen atom or a group selected from the group consistingof an alkyl group, an aryl group and a heterocyclic group (each of saidgroup may be substituted); R³, R⁴, R⁵ and R⁶ each independentlyrepresent an alkyl group that may be substituted, and R³ and R⁴, and R⁵and R⁶ may each independently bind to each other to form a ring that maybe substituted; L¹, L² and L³ each independently represent a methinegroup that may be substituted; p represents 1, 2 or 3; M represents acounter ion; and q represents a number required to neutralize the chargeof the molecule.

According to preferred embodiments of the present invention, providedare:

the compounds represented by the general formula (III) or (IV), whereinat least one of V¹, V² and V³ is a group selected from the groupconsisting of a halogen atom, an alkenyl group, an alkynyl group, anaryl group, a heterocyclic group, cyano group, an alkylthio group, anarylthio group, a heterocyclylthio group, an alkylsulfonyl group, and anarylsulfonyl group;

the compounds wherein at least one of V¹, V² and V³ is a group selectedfrom the group consisting of a halogen atom, an alkynyl group, an arylgroup and a heterocyclic group;

the compounds represented by the general formula (III) or (IV), whereinat least one of V¹, V² and V³ is an aryl group substituted with a sulfogroup or a salt thereof, a heterocyclic group substituted with a sulfogroup or a salt thereof, or an alkynyl group substituted with a sulfogroup or a salt thereof; and

the compounds represented by the general formula (III) or (IV), whereinat least one of R¹ and R² is an alkyl group or aryl group substitutedwith a reactive substituent that can form a covalent bond, inonic bondor coordinate bond with a substance to be labeled.

According to more preferred embodiments of the present invention,provided are the compounds represented by the general formula (III) or(IV) or the aforementioned preferred compounds,

wherein at least one of R¹ and R² is an alkyl group or aryl groupsubstituted with a group that can form a covalent bond with an aminogroup, hydroxyl group or thiol group of a substance to be labeled; or

wherein at least one of R¹ and R² is an alkyl group substituted with acarboxyl group.

The present invention also provides compounds represented by thefollowing general formula (V):

wherein, R¹ and R² each independently represent a hydrogen atom, or analkyl group, an aryl group, or a heterocyclic group (each of said groupmay be substituted), provided that at least one of R¹ and R² is an alkylgroup or aryl group substituted with a reactive substituent that canform a covalent bond, inonic bond, or coordinate bond with a substanceto be labeled; M represents a counter ion; q represents a numberrequired to neutralize the charge of the molecule; and W¹ and W² eachindependently represent a hydrogen atom or a group selected from thegroup consisting of a halogen atom, an alkynyl group, an aryl group, aheterocyclic group, an alkylthio group, and an arylthio group, providedthat W¹ and W² do not simultaneously represent a hydrogen atom.

The present invention further provides compounds represented by thefollowing general formula (VI):

wherein, V¹, V² and V³ each independently represent a hydrogen atom or agroup selected from the group consisting of a halogen atom, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, a heterocyclicgroup, cyano group, hydroxy group, nitro group, carboxyl group, analkoxy group, an aryloxy group, a silyloxy group, a heterocyclyloxygroup, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxygroup, an aryloxycarbonyloxy group, an amino group (including an anilinogroup), an acylamino group, an aminocarbonylamino group, analkoxycarbonylamino group, an aryloxycarbonylamino group, asulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylaminogroup, a mercapto group, an alkylthio group, an arylthio group, aheterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, anarylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, anacyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, acarbamoyl group, a phosphono group, a phosphonato group and a group thatcan form a covalent bond with a compound to be labeled (each of saidgroup may be substituted), provided that V¹, V² and V³ do notsimultaneously represent a hydrogen atom, and V¹ and V², and V² and V³may each independently form a saturated or unsaturated ring that may besubstituted; R¹ represents a hydrogen atom or a group selected from thegroup consisting of an alkyl group, an aryl group and a heterocyclicgroup (each of said group may be substituted); R³ and R⁴ represent analkyl group that may be substituted, and R³ and R⁴ may bind to eachother to form a ring that may be substituted, and compounds representedby the following general formula (VII) or salts thereof:

wherein, V¹, V² and V³ each independently represent a hydrogen atom or agroup selected from the group consisting of a halogen atom, an alkylgroup, an alkenyl group, an alkynyl group, an aryl group, a heterocyclicgroup, cyano group, hydroxy group, nitro group, carboxyl group, analkoxy group, an aryloxy group, a silyloxy group, a heterocyclyloxygroup, an acyloxy group, a carbamoyloxy group, an alkoxycarbonyloxygroup, an aryloxycarbonyloxy group, an amino group (including an anilinogroup), an acylamino group, an aminocarbonylamino group, analkoxycarbonylamino group, an aryloxycarbonylamino group, asulfamoylamino group, an alkylsulfonylamino group, an arylsulfonylaminogroup, a mercapto group, an alkylthio group, an arylthio group, aheterocyclylthio group, a sulfamoyl group, an alkylsulfinyl group, anarylsulfinyl group, an alkylsulfonyl group, an arylsulfonyl group, anacyl group, an aryloxycarbonyl group, an alkoxycarbonyl group, acarbamoyl group, a phosphono group, a phosphonato group, and a groupthat can form a covalent bond with a compound to be labeled (each ofsaid group may be substituted), provided that V¹, V² and V³ do notsimultaneously represent a hydrogen atom, and provided that V¹ and V²,and V² and V³ may each independently form a saturated or unsaturatedring that may be substituted; R¹ represents a hydrogen atom or a groupselected from the group consisting of an alkyl group, an aryl group anda heterocyclic group (each of said group may be substituted); R⁵ and R⁶represent an alkyl group that may be substituted, and R⁵ and R⁶ may bindto each other to form a ring that may be substituted.

From other aspects of the present invention, provided are afluorescence-labeling agent comprising the aforementioned compound; asubstance fluorescence-labeled with the aforementioned compound,preferably a substance for diagnosis labeled with the aforementionedcompound; an agent for diagnosis comprising a diagnostic substancefluorescence-labeled with the aforementioned compound; a method fordiagnosis comprising a use of a diagnostic substancefluorescence-labeled with the aforementioned compound; a use of theaforementioned compound for manufacture of the aforementioned agent fordiagnosis; a fluorescence contrast medium for administration into ablood vessel to visualize information in vivo, which comprises theaforementioned compound; and a use of the aforementioned compound formanufacture of the aforementioned fluorescence contrast medium.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present specification, an alkyl group may have any of straight,branched and cyclic chain or a combination thereof, and examples thereofinclude a straight or branched alkyl group having 1 to about 30 carbonatoms such as methyl group, ethyl group, n-propyl group, isopropylgroup, t-butyl group, n-octyl group, eicosyl group, and 2-ethylhexylgroup; a substituted or unsubstituted cycloalkyl group or an alkylconsisting of a combination of a cycloalkyl group and a straight orbranched alkyl group, which has 3 to 30 carbon atoms, for example,cyclohexyl group, cyclopentyl group, and 4-n-dodecylcyclohexyl group; abicycloalkyl group having 5 to 30 carbon atoms such asbicyclo[1,2,2]heptan-2-yl group and bicyclo[2,2,2]octan-3-yl group; atricycloalkyl group and the like. The same shall apply to an alkylmoiety of a substituent having the alkyl moiety (e.g., alkylthio groupand the like). Further, examples of substituted alkyl groups include,for example, 2-chloroethyl, 2-cyanoethyl and the like. A halogen atomreferred to in the specification may be any of fluorine atom, chlorineatom, bromine atom, and an iodine atom. When a functional group or aring structure is referred to as “may be substituted” (or as“substituted or unsubstituted”) in the present specification, thedescription means that the functional group or ring may have one or moresubstituents. However, types, numbers, and substituting positions of thesubstituents are not particularly limited.

As V¹, V², V³, V⁴ and V⁵ in the general formula (I), a hydrogen atom ora functional group may be used which is selected from the groupconsisting of a halogen atom, an alkyl group, an alkenyl group, analkynyl group, an aryl group, a heterocyclic group, cyano group, hydroxygroup, nitro group, carboxyl group, an alkoxy group, an aryloxy group, asilyloxy group, a heterocyclyloxy group, an acyloxy group, acarbamoyloxy group, an alkoxycarbonyloxy group, an aryloxycarbonyloxygroup, an amino group (including an anilino group), an acylamino group,an aminocarbonylamino group, an alkoxycarbonylamino group, anaryloxycarbonylamino group, a sulfamoylamino group, analkylsulfonylamino group, an arylsulfonylamino group, a mercapto group,an alkylthio group, an arylthio group, a heterocyclylthio group, asulfamoyl group, an alkylsulfinyl group, an arylsulfinyl group, analkylsulfonyl group, an arylsulfonyl group, an acyl group, anaryloxycarbonyl group, an alkoxycarbonyl group, a carbamoyl group, aphosphono group, a phosphonato group, and a group that can form acovalent bond with a compound to be labeled. These groups may have oneor more substituents at any positions, and when they have two or moresubstituents, the substituents may be the same or different. V¹, V², V³,V⁴ and V⁵ do not simultaneously represent a hydrogen atom.

Specific examples of the aforementioned groups, including those havingone or more substituents, are a halogen atom (e.g., chlorine atom,bromine atom, iodine atom and the like), an alkyl group (thoseexemplified above or the like), an alkenyl group [straight, branched, orcyclic alkenyl groups and those consisting of a combination thereof canbe used, and examples include a straight or branched alkenyl grouphaving 2 to 30 carbon atoms such as vinyl group, allyl group, prenylgroup, geranyl group, and oleyl group; a cycloalkenyl group having 3 to30 carbon atoms such as 2-cyclopenten-1-yl group and 2-cyclohexen-1-ylgroup; a bicycloalkenyl group having 5 to 30 carbon atoms such asbicyclo[2,2,1]hept-2-en-1-yl group and bicyclo[2,2,2]oct-2-en-4-ylgroup], an alkynyl group (preferably a substituted or unsubstitutedalkynyl group having 2 to 30 carbon atoms such as ethynyl group,propargyl group, trimethylsilylethynyl group, phenylethynyl group,p-sulfophenylethynyl group, p-sulfonylaminophenylethynyl group andm-sulfonylaminophenylethynyl group), an aryl group (preferably asubstituted or unsubstituted aryl group having 6 to 30 carbon atoms suchas phenyl group, p-tolyl group, naphthyl group, m-chlorophenyl group,and o-hexadecanoylaminophenyl group), a heterocyclic group (said groupcorresponds to a monovalent residue obtained by removing one hydrogenatom from an aromatic or non-aromatic heterocyclic compound, which ispreferably a 5- or 6-membered substituted or unsubstituted heterocyclicgroup, further preferably a 5- or 6-membered aromatic heterocyclic grouphaving 3 to 30 carbon atoms, such as 2-furyl group, 2-thienyl group,2-pyrimidinyl group, and 2-benzothiazolyl group), cyano group, hydroxylgroup, nitro group, carboxyl group, an alkoxy group (preferably asubstituted or unsubstituted alkoxy group having 1 to 30 carbon atomssuch as methoxy group, ethoxy group, isopropoxy group, t-butoxy group,n-octyloxy group and 2-methoxyethoxy group), an aryloxy group(preferably a substituted or unsubstituted aryloxy group having 6 to 30carbon atoms such as phenoxy group, 2-methylphenoxy group,4-t-butylphenoxy group, 3-nitrophenoxy group, and2-tetradecanoylaminophenoxy group), a silyloxy group (preferably asilyloxy group having 3 to 20 carbon atoms such as trimethylsilyloxygroup and t-butyldimethylsilyloxy group), a heterocyclyloxy group(preferably a substituted or unsubstituted heterocyclyloxy group having2 to 30 carbon atoms such as 1-phenyltetrazole-5-oxy group and2-tetrahydropyranyloxy group), an acyloxy group (preferably formyloxygroup, a substituted or unsubstituted alkylcarbonyloxy group having 2 to30 carbon atoms or a substituted or unsubstituted arylcarbonyloxy grouphaving 6 to 30 carbon atoms, for example, formyloxy group, acetyloxygroup, pivaloyloxy group, stearoyloxy group, benzoyloxy group,p-methoxyphenylcarbonyloxy group and the like), a carbamoyloxy group(preferably a substituted or unsubstituted carbamoyloxy group having 1to 30 carbon atoms such as N,N-dimethylcarbamoyloxy group,N,N-diethylcarbamoyloxy group, morpholinocarbonyloxy group,N,N-di-n-octylaminocarbonyloxy group and N-n-octylcarbamoyloxy group),an alkoxycarbonyloxy group (preferably a substituted or unsubstitutedalkoxycarbonyloxy group having 2 to 30 carbon atoms such asmethoxycarbonyloxy group, ethoxycarbonyloxy group, t-butoxycarbonyloxygroup, and n-octylcarbonyloxy group), an aryloxycarbonyloxy group(preferably a substituted or unsubstituted aryloxycarbonyloxy grouphaving 7 to 30 carbon atoms such as phenoxycarbonyloxy group,p-methoxyphenoxycarbonyloxy group and p-n-hexadecyloxyphenoxycarbonyloxygroup), an amino group (preferably an amino group, a substituted orunsubstituted alkylamino group having 1 to 30 carbon atoms, and asubstituted or unsubstituted anilino group having 6 to 30 carbon atoms,for example, amino group, methylamino group, dimethylamino group,anilino group, N-methylanilino group, diphenylamino group and the like),an acylamino group (preferably formylamino group, a substituted orunsubstituted alkylcarbonylamino group having 1 to 30 carbon atoms or asubstituted or unsubstituted arylcarbonylamino group having 6 to 30carbon atoms, for example, formylamino group, acetylamino group,pivaloylamino group, lauroylamino group, benzoylamino group,3,4,5-tri-n-octyloxyphenylcarbonylamino group and the like), anaminocarbonylamino group (preferably a substituted or unsubstitutedaminocarbonylamino group having 1 to 30 carbon atoms such ascarbamoylamino group, N,N-dimethylaminocarbonylamino group,N,N-diethylaminocarbonylamino group and morpholinocarbonylamino group),an alkoxycarbonylamino group (preferably a substituted or unsubstitutedalkoxycarbonylamino group having 2 to 30 carbon atoms such asmethoxycarbonylamino group, ethoxycarbonylamino group,t-butoxycarbonylamino group, n-octadecyloxycarbonylamino group andN-methyl-methoxycarbonylamino group), an aryloxycarbonylamino group(preferably a substituted or unsubstituted aryloxycarbonylamino grouphaving 7 to 30 carbon atoms such as phenoxycarbonylamino group,p-chlorophenoxycarbonylamino group and m-n-octyloxyphenoxycarbonylaminogroup), a sulfamoylamino groups (preferably a substituted orunsubstituted sulfamoylamino group having 0 to 30 carbon atoms such assulfamoylamino group, N,N-dimethylaminosulfonylamino group andN-n-octylaminosulfonylamino group), an alkylsulfonyl group or anarylsulfonylamino group (preferably a substituted or unsubstitutedalkylsulfonylamino group having 1 to 30 carbon atoms or a substituted orunsubstituted arylsulfonylamino group having 6 to 30 carbon atoms, forexample, methylsulfonylamino group, butylsulfonylamino group,phenylsulfonylamino group, 2,3,5-trichlorophenylsulfonylamino group,p-methylphenylsulfonylamino group and the like), a mercapto group, analkylthio group (preferably a substituted or unsubstituted alkylthiogroup having 1 to 30 carbon atoms such as methylthio group, ethylthiogroup and n-hexadecylthio group), an arylthio group (preferably asubstituted or unsubstituted arylthio group having 6 to 30 carbon atomssuch as phenylthio group, p-chlorophenylthio group andm-methoxyphenylthio group), a heterocyclylthio group (preferably asubstituted or unsubstituted heterocyclylthio group having 2 to 30carbon atoms such as 2-benzothiazolylthio group and1-phenyltetrazol-5-ylthio group), a sulfamoyl group (preferably asubstituted or unsubstituted sulfamoyl group having 0 to 30 carbon atomssuch as N-ethylsulfamoyl group, N-(3-dodecyloxypropyl)sulfamoyl group,N,N-dimethylsulfamoyl group, N-acetylsulfamoyl group, N-benzoylsulfamoylgroup and N-(N′-phenylcarbamoyl)sulfamoyl group), an alkylsulfinyl groupor an arylsulfinyl group (preferably a substituted or unsubstitutedalkylsulfinyl group having 1 to 30 carbon atoms or a substituted orunsubstituted arylsulfinyl group having 6 to 30 carbon atoms, forexample, methylsulfinyl group, ethylsulfinyl group, phenylsulfinyl groupand p-methylphenylsulfinyl group), an alkylsulfonyl group or anarylsulfonyl group (preferably a substituted or unsubstitutedalkylsulfonyl group having 1 to 30 carbon atoms or a substituted orunsubstituted arylsulfonyl group having 6 to 30 carbon atoms, forexample, methylsulfonyl group, ethylsulfonyl group, phenylsulfonylgroup, p-methylphenylsulfonyl group and the like), an acyl group(preferably formyl group, a substituted or unsubstituted alkylcarbonylgroup having 2 to 30 carbon atoms or a substituted or unsubstitutedarylcarbonyl group having 7 to 30 carbon atoms, for example, acetylgroup, pivaloyl group, 2-chloroacetyl group, stearoyl group, benzoylgroup, p-n-octyloxyphenylcarbonyl group and the like), anaryloxycarbonyl group (preferably a substituted or unsubstitutedaryloxycarbonyl group having 7 to 30 carbon atoms such asphenoxycarbonyl group, o-chlorophenoxycarbonyl group,m-nitrophenoxycarbonyl group and p-t-butylphenoxycarbonyl group), analkoxycarbonyl group (preferably a substituted or unsubstitutedalkoxycarbonyl group having 2 to 30 carbon atoms such as methoxycarbonylgroup, ethoxycarbonyl group, t-butoxycarbonyl group andn-octadecyloxycarbonyl group), a carbamoyl group (preferably asubstituted or unsubstituted carbamoyl group having 1 to 30 carbon atomssuch as carbamoyl group, N-methylcarbamoyl group, N,N-dimethylcarbamoylgroup, N,N-di-n-octylcarbamoyl group and N-(methylsulfonyl)carbamoylgroup), a phosphono group, a phosphonato group, and a group that canform a covalent bond with a substance to be labeled (e.g., anisothiocyanato group an isocyanato group, a succinimidyl ester group, ahalogen-substituted triazinyl group, a halogen-substituted pyrimidinylgroup, a sulfonyl halide group, an α-haloacetyl group, a maleimidylgroup, an aziridinyl group and the like).

When the aforementioned functional groups is substituted, theaforementioned groups or sulfonic acid group or a salt thereof can beused as the substituent. Examples of the substituent on theaforementioned functional groups include a sulfonic acid group or a saltthereof, as well as an alkylcarbonylaminosulfonyl group, anarylcarbonylaminosulfonyl group, an alkylsulfonylaminocarbonyl group, anarylsulfonylaminocarbonyl group and the like. More specifically,examples of substituents on the aforementioned functional groups includea sulfonic acid group or a salt thereof, methylsulfonylaminocarbonylgroup, p-methylphenylsulfonylaminocarbonyl group, acetylaminosulfonylgroup, benzoylaminosulfonyl group and the like, and a sulfonic acidgroup or a salt thereof is preferred as the substituent.

V¹ and V², V² and V³, and V⁴ and V⁵ may bind to each other to form asaturated or unsaturated ring. As the ring thus formed, a 5-, 6- or7-membered ring is preferred. Further, the ring may contain one or morehetero atoms (a hetero atom referred to in the specification means, forexample, an oxygen atom, a nitrogen atom, a sulfur atom, a metal atom orthe like), and when two or more hetero atoms are contained, they may bethe same or different. Further, one or more substituents such as thoseexplained as for V¹ and others may exist at any position on the ringformed, and when two or more substituents exist, they may be the same ordifferent.

Preferred as V¹, V², V³, V⁴ and V⁵ are a halogen atom, an alkyl grouphaving 1 to 6 carbon atoms, an alkenyl group having 2 to 30 carbonatoms, an alkynyl group having 2 to 30 carbon atoms, an aryl grouphaving 6 to 20 carbon atoms, a heterocyclic group (a 5- or 6-memberedaromatic or non-aromatic substituted or unsubstituted heterocyclicgroup, preferably a 5- or 6-membered aromatic heterocyclic having 3 to30 carbon atoms), an alkylthio group having 1 to 30 carbon atoms, anarylthio group having 6 to 30 carbon atoms, a substituted orunsubstituted heterocyclylthio group having 2 to 30 carbon atoms, asulfamoyl group having 0 to 30 carbon atoms, an alkylsulfonyl grouphaving 1 to 30 carbon atoms and an arylsulfonyl group having 6 to 30carbon atoms. An isothiocyanato group, an isocyanato group, asuccinimidyl ester group, a halogen-substituted triazinyl group, ahalogen-substituted pyrimidinyl group, a sulfonyl halide group, anα-haloacetyl group, a maleimidyl group and an aziridinyl group, whichcan form a covalent bond with a substance to be labeled, are alsopreferred.

Further preferred as V¹, V², V³, V⁴ and V⁵ are a halogen atom, an alkylgroup having 1 to 6 carbon atoms, an alkynyl group having 2 to 30 carbonatoms, an aryl group having 6 to 20 carbon atoms, a heterocyclic group,an alkylthio group having 1 to 30 carbon atoms, an arylthio group having6 to 30 carbon atoms, a substituted or unsubstituted heterocyclylthiogroup having 2 to 30 carbon atoms, and the group that can form acovalent bond with a substance to be labeled, which is explained above.

As R¹ in the general formula (I), for example, a hydrogen atom, an alkylgroup having 1 to 20 carbon atoms (e.g., methyl group, ethyl group,propyl group, butyl group, cyclohexyl group and the like), an aryl grouphaving 6 to 20 carbon atoms (e.g., phenyl group, tolyl group, naphthylgroup and the like) and a heterocyclic group (e.g., 2-pyridyl group,4-pyridyl group, pyrazolyl group, triazolyl group, tetrazolyl group andthe like) can be used. The aforementioned alkyl group, aryl group, andheterocyclic group may have one or more substituents at any positions.Types of the substituents are not particularly limited, and the groupsexplained as for the aforementioned V¹ or others and a sulfonic acidgroup or a salt thereof, as well as a reactive substituent for labelinga substance to be labeled through a covalent bond, an ionic bond, ahydrogen bond or the like may exist.

Examples of the substance to be labeled include antibody, protein,peptide, enzyme substrate, hormone, lymphokine, metabolic product,receptor, antigen, hapten, lectin, avidin, streptavidin, toxin,carbohydrate, polysaccharide, nucleic acid, deoxynucleic acid, nucleicacid derivatives, deoxynucleic acid derivatives, DNA fragment, RNAfragment, DNA fragment derivatives, RNA fragment derivatives,naturally-derived drug, virus particle, bacterium particle, viruscomponent, yeast component, blood cell, blood cell component, bacterium,bacterial component, natural or synthetic lipid, drug, poison,environmental pollutant, polymer, polymer particle, glass particle,plastic particle, polymer membrane and the like. Examples of thereactive substituent for labeling these substances through a covalentbond, ionic bond or hydrogen bond include, for example, a succinimidylester group, a halogen-substituted triazinyl group, ahalogen-substituted pyrimidinyl group, a sulfonyl halide group, anα-haloacetyl group, a maleimidyl group, an aziridinyl group and thelike.

R¹ is preferably a substituted or unsubstituted alkyl group having 1 to20 carbon atoms, more preferably a substituted alkyl group having 1 to20 carbon atoms. Examples of preferred substituents on the alkyl groupinclude the aforementioned reactive substituents, as well as carboxylgroup, an amino group, hydroxy group, a mercapto group, a sulfonic acidgroup or a salt thereof, an alkylamido group (an alkyl moiety of saidalkylamido group may have one or more substituents selected from thegroup consisting of the aforementioned reactive substituents, carboxylgroup, an amino group, hydroxy group and a mercapto group) and the like.

In the general formula (I), Q represents a group of atoms required toform a methine dye chromophore. Types of the methine dye are notparticularly limited. Preferred methine dyes include, for example,cyanine dyes, merocyanine dyes, rhodacyanine dyes, trinucleousmerocyanine dyes, tetranucleous merocyanine dyes, allopolar dyes, styryldyes, styryl base dyes, hemicyanine dyes, streptocyanine dyes,hemioxonole dyes and the like, more preferred dyes include cyanine dyes,merocyanine dyes and rhodacyanine dyes, and most preferred dyes includecyanine dyes (as for their charged state, they may be in any form ofcation, anion, or betaine). Details of these dyes are described in F. M.Harmer, “Heterocyclic Compounds—Cyanine Dyes and Related Compounds”,John Wiley and Sons, New York, London, 1964; D. M. Sturmer,“Heterocyclic Compounds—Special Topics in Heterocyclic Chemistry”,Chapter 18, Section 14, 482 to 515 pages, John Wiley and Sons, New York,London, 1977 and the like.

As the cyanine dyes, merocyanine dyes, and rhodacyanine dyes, thosedisclosed in U.S. Pat. No. 5,340,694, pages 21 and 22, (XI), (XII) and(XIII) are preferred. More preferred are those represented by thegeneral formula (I) of the present invention wherein cyanine dyestructure of the formula (II) is formed.

In the general formula (II), V¹, V², V³, V⁴, V⁵, V⁶, V⁷, V⁸, V⁹ and V¹⁰have the same meanings as V¹ mentioned in the general formula (I). V¹ toV¹⁰ are independent from one another, however, V¹, V², V³, V⁴ and V⁵ donot simultaneously represent a hydrogen atom. R¹ and R² are independentfrom each other, and they have the same meaning as R¹ mentioned in thegeneral formula (I). R³, R⁴, R⁵ and R⁶ are independent from one another,and they have the same meaning as R³ and R⁴ mentioned in the generalformula (I).

In the general formula (II), L¹, L² and L³ represent a substituted orunsubstituted methine group. Examples of the substituents existing onthe methine group include a substituted or unsubstituted alkyl grouphaving 1 to 15 carbon atoms, preferably 1 to 10 carbon atoms, mostpreferably 1 to 5 carbon atoms (e.g., methyl group, ethyl group,carboxyethyl group and the like), a substituted or unsubstituted arylgroup having 6 to 30 carbon atoms, preferably 6 to 20 carbon atoms, mostpreferably 6 to 15 carbon atoms (e.g., phenyl group, o-carboxyphenylgroup and the like), a substituted or unsubstituted heterocyclic grouphaving 3 to 20 carbon atoms, preferably 4 to 15 carbon atoms, mostpreferably 6 to 10 carbon atoms (e.g., N,N-dimethylbarbituric acid groupand the like), a halogen atom (e.g., fluorine atom, chlorine atom,bromine atom, iodine atom and the like), an alkoxy group having 1 to 20carbon atoms, preferably 1 to 15 carbon atoms, most preferably 1 to 10carbon atoms (e.g., methoxy group, ethoxy group and the like), an aminogroup having 0 to 20 carbon atoms, preferably 2 to 15 carbon atoms, mostpreferably 4 to 15 carbon atoms (e.g., methylamino group, dimethylaminogroup, N-methyl-N-phenylamino group, N-methylpiperazino group and thelike), an alkylthio group having 1 to 15 carbon atoms, preferably 1 to10 carbon atoms, most preferably 1 to 8 carbon atoms (e.g., methylthiogroup, ethylthio group and the like), an arylthio groups having 6 to 20carbon atoms, preferably 6 to 18 carbon atoms, most preferably 6 to 15carbon atoms (e.g., phenylthio group, p-methylthio group and the like).

When the compounds of the present invention are used for diagnosticpurpose, it is preferred that the aforementioned substituents arefurther substituted with a reactive group such as a succinimidyl estergroup, a halogen-substituted triazinyl group, a halogen-substitutedpyrimidinyl group, a sulfonyl halide group, an α-haloacetyl group, amaleimidyl group and an aziridinyl group. Symbol “p” represents 1, 2 or3. Symbol “p” is preferably 1 or 2, and p is most preferably 1 whichcorresponds to the structure represented by the general formula (V).

M represents a counter ion. M may be a cation or an anion, and examplesof the cation include an alkali metal ion such as sodium ion, potassiumion and lithium ion and an organic ion such as tetraalkylammonium ionand pyridinium ion. The anion may be an inorganic anion or an organicanion. Examples of the anion include a halogen anion (e.g., fluorineion, chlorine ion, bromine ion, iodine ion), a substituted arylsulfonateion (e.g., p-toluenesulfonate ion, p-chlorobenzenesulfonate ion), anaryldisulfonate ion (e.g., 1,3-benzenedisulfonate ion,1,5-naphthalenedisulfonate ion), an alkylsulfate ion (e.g.,methylsulfate ion), sulfate ion, thiocyanate ion, perchlorate ion,tetrafluoroborate ion, picrate ion, acetate ion,trifluoromethanesulfonate ion and the like. M may be a hydrogen ion.Preferred counter ions are an ammonium ion, an alkali metal ion, ahalogen anion and a substituted arylsulfonate ion, and more preferredare an alkali metal ion, a halogen anion and a substituted arylsulfonateion. Symbol “q” represents a number required to neutralize charge of themolecule.

In the general formula (III), V¹, V², V³, R¹, R³, R⁴ and Q have the samemeanings as those of the general formula (I), respectively. In thegeneral formula (IV), V¹, V², V³, V⁶, V⁷, V⁸, R¹, R², R³, R⁴, R⁵, R⁶,L¹, L², L³, M, p and q have the same meanings as those of the generalformula (II), respectively. In the general formula (VI), V¹, V², V³, R¹,R³, R⁴, M and q have the same meanings as those of the general formula(II), respectively. In the general formula (VII), V¹, V², V³, R³, and R⁴have the same meanings as those of the general formula (II),respectively.

In the general formula (V), R¹, R², M and q have the same meanings asthose of the general formula (II), respectively. The halogen atom,alkynyl group, aryl group, heterocyclic group, alkylthio group andarylthio group represented by W¹ and W² have the same meanings as thegroups explained as for V¹ and others in the formula (I), respectively.It is preferred that at least one acid group (e.g., a sulfonic acidgroup or a salt thereof, carboxylic acid group, phosphoric acid groupand the like) is introduced on each of the aforementioned substituentsexcept for the halogen. The acid group may bind to the aforementionedalkynyl group, aryl group, heterocyclic group, alkylthio group orarylthio group via a bridging group such as an alkylene group, however,it is preferred that the acid group directly binds to the aforementionedsubstituents.

The compounds of the present invention may exist as acid addition salts,and they may exist as base addition salts depending on types ofsubstituents. Examples of the salts include mineral acid salts such ashydrochloride, sulfate, nitrate and phosphate, organic acid salts suchas methanesulfonate, p-toluenesulfonate, tartrate, citrate and maleate,metal salts such as sodium salt, potassium salt and calcium salt,ammonium salts, organic amine salts such as triethylamine salt, aminoacid salts such as and glycine salt and the like. Further, the compoundsof the present invention may exist as hydrates or solvates, and each ofthese substances also falls within the scope of the present invention.Further, the compounds of the present invention may have one or moreasymmetric carbons depending on types of the substituents, and any ofstereoisomers such as optical isomers and diastereoisomers, mixtures ofstereoisomers, racemates and the like also falls within the scope of thepresent invention.

Preferred examples of the compounds of the present invention representedby the aforementioned general formulas are shown below. However, thescope of the present invention is not limited to the following specificcompounds.

No. V¹ V⁷ R¹ R² M I-11

—(CH₂)₅—COOH 2K⁺ I-12

—(CH₂)₅—COOH 2K⁺ I-13

2K⁺ I-14

2K⁺ I-15

4Na⁺ I-16

—(CH₂)₅—COOH — I-17

—(CH₂)₅—COOH 3K⁺ I-18

—(CH₂)₅—CONH—(CH₂)₂COOH — I-19

—(CH₂)₅—COOH K⁺ I-20

—(CH₂)₅—COOH K⁺ I-21

3K⁺ I-22

—(CH₂)₅—COOH — I-23

—(CH₂)₄SO₃ ⁻ —(CH₂)₅—COOH K⁺ I-24

H —(CH₂)₄SO₃ ⁻ —(CH₂)₅—COOH — I-25

Na⁺ I-26

Na⁺ I-27

—(CH₂)₅—COOH 2K⁺ I-28

—(CH₂)₅—COOH 2K⁺ I-29

—(CH₂)₅—COOH 4K⁺ I-30

2I⁻

No. V R¹ R² M I-31 —CF₃ —CH₃ —(CH₂)₅—COO⁻ — I-32 —CN

—(CH₂)₅—COOH — I-33 —OH —(CH₂)₃—SO₃ ⁻

— I-34 —NO₂ —(CH₂)₃—SO₃ ⁻

— I-35 —COOH —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—NCS — I-36 —OCH₃

K⁺ I-37

—(CH₂)₅—COOH — I-38

—CH₃ —(CH₂)₅—COO⁻ — I-39

—CH₃

I⁻ I-40 —OCOCH₃ —CH₃

— I-41 —OCONH₂ —CH₃ —(CH₂)₅—COO⁻ — I-42 —OCOOCH₃ —(CH₂)₃—SO₃ ⁻—(CH₂)₅—COOH — I-43

—(CH₂)₃—SO₃ ⁻ —(CH₂)₅—NCS 2Na⁺ I-44 —N(CH₃)₂ —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—NCS —I-45 —NHCO(CH₂)COOH —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—SO₃ ⁻ K⁺ I-46 —NHCONH₂

—(CH₂)₅—COOH — I-47 —NHCOOCH₃ —CH₃ —(CH₂)₅—COO⁻ — I-48 —NHSO₂NH₂ —CH₃—(CH₂)₅—COO⁻ — I-49 —NHSO₂CH₃

—(CH₂)₅—NCS K⁺ I-50

— I-51

—CH₃ —(CH₂)₅—COO⁻ — I-52 —SO₂NH₂ —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—COOH — I-53—SOCH₃ —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—COOH — I-54 —SO₂CH₃ —(CH₂)₃—SO₃ ⁻—(CH₂)₅—COOH — I-55 —COCH₃ —CH₃ —(CH₂)₅—COO⁻ — I-56 —COOC₂H₅ —CH₃—(CH₂)₅—COO⁻ — I-57

—CH₃ —(CH₂)₅—COO⁻ 2K⁺ I-58 —CONH(CH₂)₂COOH —CH₃ —(CH₂)₅—COO⁻ — I-59 —PO₃⁻⁻ —(CH₂)₃—SO₃ ⁻ —(CH₂)₅—COOH 4Na⁺ I-60

I-61

I-62

I-63

I-64

I-65

I-66

I-67

I-68

I-69

I-70

I-71

I-72

I-73

I-74

No. R¹ M VI-1 —CH₃ — VI-2

VI-3 —(CH₂)₂SO₃ ⁻

VI-4 —(CH₂)₄SO₃ ⁻

VI-5

VI-6

VI-7 —(CH₂)₅—COOC₂H₅ — VI-8 —(CH₂)₁₁—COOCH₃ —

No. R¹ W M VI-9

H — VI-10 —(CH₂)₂SO₃ ⁻ H — VI-11

H Na⁺ VI-12 —(CH₂)₅—COOC₂H₅ H Br⁻ VI-13

—(CH₂)₅—COOC₂H₅ — VI-14 —(CH₂)₂SO₃ ⁻ —(CH₂)₅—COOC₂H₅ — VI-15

—(CH₂)₅—COOC₂H₅ Na⁺

No. R¹ M VI-16

— VI-17 —(CH₂)₅COOC₂H₅ Br⁻

No. R¹ M VI-18

— VI-19

K⁺ VI-20

Na⁺ VI-21 —(CH₂)₅COOC₂H₅ Br⁻

No. W R¹ M VI-22

— VI-23

—(CH₂)₅COOC₂H₅ Br⁻ VI-24

Na⁺ VI-25

—(CH₂)₅COOC₂H₅ — VI-26

Na⁺ VI-27

Na⁺ VI-28

—(CH₂)₃SO₃ ⁻ —

No. R¹ W M VI-29

H — VI-30 —(CH₂)₂SO₃ ⁻ H — VI-31

H Na⁺ VI-32 —(CH₂)₅—COOC₂H₅ H Br⁻ VI-33

—(CH₂)₅—COOC₂H₅ — VI-34 —(CH₂)₂SO₃ ⁻ —(CH₂)₅—COOC₂H₅ — VI-35

—(CH₂)₅—COOC₂H₅ Na⁺

No. W VII-1

VII-2

VII-3

VII-4

VII-5

VII-6

VII-7

VII-8

VII-9

VII-10

VII-11

VII-12

VII-13 —CF₃ VII-14 —CN VII-15 —OH VII-16 —NO₂ VII-17 —COOH VII-18 —OCH₃VII-19

VII-20

VII-21

VII-22 —OCOCH₃ VII-23 —COCNH₂ VII-24 —OCOOCH₃ VII-25

VII-26 —N(CH₃)₂ VII-27 —NHCO(CH₂)COOH VII-28 —NHCONH₂ VII-29 —NHCOOCH₃VII-30 —NHSO₂NH₂ VII-31 —NHSO₂CH₃ VII-32

VII-33

VII-34 —SO₂NH₂ VII-35 —SOCH₃ VII-36 —SO₂CH₃ VII-37 —COCH₃ VII-38—COOC₂H₅ VII-39

VII-40 —CONH(CH₂)₂COOH VII-41 —PO⁻⁻ VII-42 —Cl VII-43 —Br VII-44

VII-45

VII-46

VII-47

VII-48

VII-49

VII-50

VII-51

VII-52

VII-53

VII-54

VII-55

VII-56

VII-57

VII-58

VII-59

VII-60

VII-61

VII-62

VII-63

VII-64

VII-65

VII-66

VII-67

VII-68

VII-69

VII-70

VII-71

VII-72

VII-73

VII-74

VII-75

VII-76

VII-77

VII-78

VII-79

VII-80

VII-81

VII-82

VII-83

VII-84

VII-85

VII-86

VII-87

VII-88

VII-89

VII-90

VII-91

VII-92

VII-93

VII-94

VII-95

VII-96

VII-97

VII-98

VII-99

VII-100

VII-101

VII-102

The compounds of the present invention represented by the aforementionedgeneral formulas are useful as fluorescence-labeling agents. Substancesthat can be labeled with the compounds of the present invention are notparticularly limited, and any substances can be used as an object of thelabeling, which include low molecular compounds, high molecularcompounds, organic compounds, inorganic compounds, biologicalsubstances, natural organic compounds, microorganisms and the like. Morespecifically, examples of the substances to be labeled include antibody,protein, peptide, enzyme substrate, hormone, lymphokine, metabolicproduct, receptor, antigen, hapten, lectin, avidin, streptavidin, toxin,carbohydrate, polysaccharide, nucleic acid, deoxynucleic acid, nucleicacid derivative, deoxynucleic acid derivative, DNA fragment, RNAfragment, DNA fragment derivative, RNA fragment derivative,naturally-derived drug, virus particle, bacterium particle, viruscomponent, yeast component, blood cell, blood cell component, bacterium,bacterial component, natural or synthetic lipid, synthetic drug, poison,environmental pollutant, polymer, polymer particle, glass particle,plastic particle, polymer membrane and the like.

Among them, preferred substances to be labeled are antibody, protein,peptide, nucleotide, hormone, saccharide, lipid, vitamin, alkaloid,antibiotic and the like. Specific examples of the protein and peptideinclude, for example, immunoglobulins such as IgG, IgA, IgM, IgD andIgE, monoclonal antibodies directed to various proteins or membraneantigens of leucocyte, enzymes such as peroxidase, glucose oxidase, andalkaline phosphatase, and specific examples of the nucleotide include,for example, DNA, RNA, synthetic oligonucleotides, syntheticpolynucleotides, ATP, CPT, GTP, TTP, UTP, dATP, dCTP, dGTP, dTTP, dUTP,ddATP, ddCTP, ddGTP, ddTTP, ddUTP, derivatives thereof. Specificexamples of the saccharide include, for example, polysaccharides such asglycogen, starch and mannan, oligosaccharides and monosaccharides suchas glucose and mannose, and specific examples of the lipid include, forexample, phosphatidylcholine, phosphatidylethanolamine, fats, aliphaticacids. Examples of the hormone include, for example, peptide hormonessuch as insulin, growth hormones, epidermal growth factor, oxytocin,vasopressin and secretin, steroid hormones such as androgen andestrogen, catecholamines such as adrenaline and noradrenaline, andspecific examples of the vitamin include, for example, vitamin A,vitamin B (B1, B2, B6, B12 and the like), vitamin C, vitamin D, vitaminE, biotin, folic acid. Specific examples of the alkaloid include, forexample, opium alkaloids, tropane alkaloids such as atropine, indolealkaloids such as vinblastine, isoquinoline alkaloids such as coptisrhizome, and specific examples of the antibiotic include penicillin,cephalosporin, kanamycin, erythromycin. Among these substances to belabeled, substances that can be used for diagnosis (referred to as“diagnostic substances” in the specification, e.g., antibodies,proteins, peptides and the like) are preferred substances to be labeled.

Various techniques for introducing a fluorescence-labeling agent into asubstance to be labeled are known, and when the compounds of the presentinvention are used as fluorescence-labeling agents, it is possible toappropriately select and use any means available to those skilled in theart. For example, a functional group in a substance to be labeled suchas amino group and hydroxyl group can be directly bound to a reactivesubstituent such as carboxyl group and active ester group in thecompounds of the present invention through an ionic bond or covalentbond, or after chemical modification such as introduction of a linkerinto a part of a substance to be labeled, the resulting product can bereacted with the compounds of the present invention. Alternatively, itis also possible to label a single-stranded DNA or RNA via a hydrogenbond with a base of a nucleic acid, or by intercalation intodouble-stranded DNA. The labeled substance after the reaction can bepurified by commonly used separation techniques such as chromatography,electrophoresis, recrystallization and washing.

When the compounds of the present invention are used for DNA analysis,the compounds of the present invention can be incorporated into a probeor primer, for example, according to the method described by Ruth (JerryL. Ruth, DNA, 3, 128 (1984)). The method of reacting a hydrophilicpolyfunctional polymer such as proteins with a dye compound having areactive substituent (e.g., active ester, isothiocyanate, iodoacetyl andthe like) for labeling can be readily performed, for example, accordingto the method disclosed in U.S. Pat. No. 5,569,587. A dye having acarboxyl group that is not converted to an active ester can be labeled,for example, by the method described in Japanese Patent UnexaminedPublication No. 6-222059.

Further, for example, an anti-tumor antibody can be labeled with thecompound of the present invention and the labeled antibody can bebrought into contact with a tissue or organ to prove existence of cancercell or cancer tissue. For diagnosis, a tissue slice can be fixed by anappropriate method such as the paraffin method and observed under amicroscope, or a tissue in vivo can be immunochemically stained andobserved by using an endoscope. Recently, various fluorescence imagingmethods using near infrared-ray fluorescent substances have beenproposed (e.g., Japanese Patent Unexamined Publication No. 9-309845; J.Neurosurg., 87, pp. 738-745, 1997; Iyoh Denshi to Seitai Kogaku(Electrons for Medical Use and Bioengineering), 34, pp.316-322, 1996 andthe like), and the compounds of the present invention can be used asdiagnostic agents utilizing such fluorescence imaging methods.

When diagnostic substances labeled with the compounds of the presentinvention (e.g., labeled antibodies and the like) are used as diagnosticagents, they are preferably prepared in the form of a pharmaceuticalcomposition by using one or more kinds of additives for pharmaceuticalpreparations. For example, a pharmaceutical composition in the form ofsolid, solution or the like can be prepared by using suitable additivesfor pharmaceutical preparations such as buffering agents, dissolvingaids, pH modifiers, excipients, and preservatives. Those skilled in theart can appropriately choose a form of a pharmaceutical compositionsuitable for diagnosis or treatment as well as a method for preparationthereof. Furthermore, it is also possible to use the compounds of thepresent invention as fluorescence contrast media, which are administeredinto a blood vessel to visualize information in vivo (e.g., existence ofcancer tissue and the like).

Methods for preparing the compounds of the present invention are notparticularly limited, and they can be prepared via various syntheticroutes. The compounds represented by the general formula (VII) can beprepared by applying a reaction well known as the Fischer's indolesynthesis. The Fischer's indole synthesis is described in variouspublications. As for reaction conditions (temperature, solvent, time,reaction agent and the lie), R. J. Sudborg, “The Chemistry of Indoles”,Academic Press, New York, 1970 and the reference cited therein can bereferred to. For preparation of the compounds of the present invention,a higher reaction temperature is often required compared with usualindole or indolenine synthesis, however, reaction temperature can beappropriately chosen by those skilled in the art.

For preparation of the compounds represented by the general formula(VII), a coupling reaction of an aromatic compound or acetylene can beapplied to an indolenine introduced with a leaving group such as ahalogen. As for coupling reaction, “Cross-Coupling Reaction of OrganicBoron Compound Using Palladium Catalyst” described in Chemical Review,vol. 95, p.2457 (1995) and references cited therein can be referred to,and the reaction known as Heck reaction described in Journal of OrganicChemistry, vol. 55, p.63 (1990) can be applied to easily produce atarget substance. A compound represented by the general formula (VI) canbe synthesized by treating a compound represented by the general formula(VII) with any of various alkylating agents.

Dyes represented by the general formulas (I) to (V) can be synthesizedfrom a compound represented by the general formula (VI) by using a knownmethod. As for reaction conditions (temperature, solvent, time, reactionagents and the like), explanations are given in F. M. Harmer,“Heterocyclic Compounds—Cyanine Dyes and Related Compounds”, John Wileyand Sons, New York, London, 1964; D. M. Sturmer, “HeterocyclicCompounds—Special Topics in Heterocyclic Chemistry”, Chapter 18, Section14, 482 to 515 pages and the like.

Methods for preparation of typical compounds are specifically describedin the examples of the specification. Accordingly, those skilled in theart can prepare any compound falling within the scope of theaforementioned general formulas by referring to the specificexplanations in the following example and appropriately choosing acompound as a starting material, reaction conditions, reagents and thelike, and applying modification or alteration to the methods describedin the examples as required. However, methods for preparing thecompounds of the present invention represented by the aforementionedgeneral formulas are not particularly limited, and it should beunderstood that those prepared by any methods fall within the scope ofthe present invention.

EXAMPLES Example 1 Synthesis of Compound VII-43

5-Bromo-2-hydrazinopyridine (10 g) and 3-methyl-2-butanone (20 ml) weremixed and allowed to react at 80° C. for 15 minutes. After evaporationof excess 3-methyl-2-butanone, the reaction mixture was added with1,4-butanediol (15 ml) and allowed to react for 5 hours with heating at230° C. The reaction mixture was cooled and subjected to silica gelcolumn chromatography without treatment. The solvent was evaporated fromthe fraction containing the target substance, and then the residue wascrystallized from hexene/ethyl acetate to obtain Compound VII-43 aslight brown crystals.

Yield: 2.0 g (15.7%) Melting point: 132-134° C. H-NMR (CDCl₃), δ 8.50(d, 1H), 7.70 (d, 1H), 2.40 (s, 3H), 1.86 (s, 6H)

Example 2 Syntheses of Compounds VII-13 to VII-31 and Compounds VII-34and VII-35

Compounds VII-13 to VII-31 and Compounds VII-34 and VII-35 weresynthesized from corresponding hydrazinopyridines under the sameconditions as those for Compound VII-43 though they were obtained in alow yield. The structures of the compounds were verified based on valuesobtained in mass spectrometry and elemental analysis.

Example 3 Synthesis of Compound VII-1

Compound VII-1 was synthesized by the synthetic route descried below.

(1) Synthesis of Compound VII-1b

Compound VII-1a (121 g) was suspended in acetic acid (3 L) and addeddropwise with bromine (94 g) under water cooling over 2 hours. Thereaction mixture was further stirred for 1 hour, and then the crystalswere collected by filtration to obtain Compound VII-1b. Yield: 182.3 g(62%). H-NMR (D₂O), δ 7.83 (d, 2H), 7.70 (d, 2H), 5.42 (t, 1H), 4.20 (d,2H)

(2) Synthesis of Compound VII-1c

Compound VII-1b (100 g) was added to 20% aqueous potassium hydroxide andthe mixture was allowed to react at 90° C. One hour after the reactionwas started, the reaction mixture became uniform. The reaction wasfurther continued for 2 hours. Then, after the reaction mixture wasstirred with ice cooling for 1 hour, crystals of the target substanceCompound VII-1c deposited. Yield: 41 g (68%). H-NMR (DMSO-d₆) δ 7.61 (d,2H), 7.43 (d, 2H), 4.20 (s, 1H)

(3) Synthesis of Compound VII-1

Compound VII-43 (8.3 g) and Compound VII-1c (5.0 g) were dissolved inDMF (50 ml) under a nitrogen flow, added with potassium carbonate (6.2g) and tetrakistriphenylphosphine palladium (0.52 g), and the mixturewas allowed to react at 90° C. for 6 hours. When ethylene acetate wasadded to the reaction mixture, crystals containing mineral saltprecipitated, and then the crystals were collected by filtration. Whenthe crystals were added with water (200 ml), chloroform (200 ml),hydrochloric acid (5 ml) and tributylamine (10 ml) for separation, thetarget substance was extracted in the chloroform layer as tributylaminesalt. The chloroform layer was washed twice with water and thenconcentrated. The residue was crystallized from ethyl acetate/hexane toobtain Compound II-1. Yield: 7.0 g (89%). H-NMR (DMSO-d₆) δ 8.50 (d,1H), 8.05 (d, 1H), 7.60 (d, 2H), 7.45 (d, 2H), 2.35 (s, 3H), 1.35 (s,6H)

Example 4 Syntheses of Compound VII 2 and Compound VII-3

Compounds VII-2 and VII-3 were synthesized by the synthetic routedescribed below.

(1) Synthesis of Compound VII-2b

Compound VII-2a (11.7 g) was dissolved in a two-layer system of ethylacetate (100 ml) and aqueous sodium hydrogencarbonate (100 ml), andadded with pyridine (8 ml). The mixture was then added dropwise withmethanesulfonyl chloride (22 g), and allowed to react for 8 hours underice cooling. The ethyl acetate layer was separated and washed withsaturated aqueous sodium hydrogencarbonate. The ethyl acetate wasevaporated and the reside was recrystallized from a small amount ofethyl acetate to obtain Compound VII-2b. Yield: 16.6 g (85%). H-NMR(DMSO-d₆) δ 10.02 (s, 1H), 7.41 (d, 2H), 7.18 (d, 2H), 4.10 (s, 1H),3.08 (s, 3H)

(2) Synthesis of Compound VII-2

Compound VII-48 (2.6 g) and Compound VII-2b (3.0 g) were dissolved inDMF (40 ml) under a nitrogen flow, and the mixture was added withpotassium carbonate (4.1 g) and tetrakistriphenylphosphine palladium(0.38 g), and then allowed to react at 90° C. for 2.5 hours. Thereaction mixture was extracted with chloroform (200 ml), and the extractwas concentrated. The residue was crystallized from ethyl acetate/hexaneto obtain crude crystals of Compound VII-2. The crude crystals werefurther recrystallized from ethyl acetate to obtain Compound VII-2.Yield: 3.5 g (91%). H-NMR (DMSO-d₆) δ 9.92 (s, 1H), 8.50 (d, 1H), 8.05(d, 1H), 7.52 (d, 2H), 7.25 (d, 2H), 3.10 (s, 3H), 2.35 (s, 3H), 1.35(s, 6).

(3) Synthesis of Compound VII-8

Compound VII-2 (0.51 g) was dissolved in DMF (7 ml), and the solutionwas added with ethyl 6-bromohexanoate (0.32 g) and potassium carbonate(0.60 g) and then the mixture was allowed to react at 55° C. for 5hours. The reaction mixture was extracted with ethyl acetate, and theextract was concentrated and then subjected to silica gel columnchromatography to obtain Compound VII-3. Yield: 0.46 g (64%). H-NMR(CDCl₃) δ 8.52 (d, 1H), 8.05 (d, 1H), 7.58 (d, 2H), 7.42 (d, 2H), 4.02(dd, 2H), 3.65 (m, 2H), 3.00 (s, 3H), 2.35 (s, 3H), 2.20 (t, 2H),1.50-1.30 (m, 12H), 1.18 (t, 3H)

Example 5 Syntheses of Compound VII-4 and Compound VII-5

Compound VII-4 and Compound VII-5 were obtained by usingm-aminophenylacetylene as a starting material with the syntheticconditions for Compound VII-2 and Compound VII-3.

Compound VII-4: H-NMR (DMSO-d₆) δ 9.92 (s, 1H), 8.50 (d, 1H), 8.05 (d,1H), 7.42-7.20 (m, 4H), 3.00 (s, 3H), 2.30 (s, 3H), 1.25 (s, 6H)

Compound VII-5: H-NMR (CDCl₃) δ 8.52 (d, 1H), 8.05 (d, 1H), 7.38-7.10(m, 4H), 4.00 (dd, 2H), 3.60 (m, 2H), 2.98 (s, 3H), 2.30 (s, 3H), 2.20(t, 2H), 1.50-1.30 (m, 12H), 1.18 (t,3H).

Example 6 Synthesis of Compound VII-6

Compound VII-43 (2.4 g), potassium carbonate (3.5 g), triphenylphosphine(0.31 g) and cuprous iodide (0.12 g) were added to diethylene glycoldimethyl ether (20 ml) and water (20 ml) under a nitrogen flow, and thenthe mixture was stirred at 25° C. for 30 minutes. Then, the reactionmixture was added and propargyl alcohol (1.5 ml). The reactiontemperature was raised to 80° C., and the reaction mixture was allowedfor 16 hours at the same temperature. The reaction mixture was filteredthrough Cerite, and the filtrate was made acidic with addition ofhydrochloride acid and washed with ethyl acetate. When the acidicaqueous layer was made to be pH 8.0 with addition of aqueous sodiumhydroxide and the aqueous layer was mixed with chloroform and separated,the target substance was extracted in the chloroform layer. Thechloroform was evaporated and the residue was recrystallized from ethylacetate to obtain Compound VII-6. Yield: 1.7 g (80%) H-NMR (DMSO-d₆) δ8.52 (d, 1H), 7.62 (d, 1H), 4.52 (m, 2H), 2.33 (s, 3H), 1.30 (s, 6H)

Example 7 Synthesis of Compound VII-7

A reaction was performed by using 2-methyl-3-butyn-2-ol instead ofpropargyl alcohol used in the synthesis of Compound VII-6 under the sameconditions as the synthesis of Compound VII-6 to synthesize CompoundVII-7. The structure was verified by mass spectrometry.

Example 8 Synthesis of Compound VII-8

Compound VII-43 (1.2 g) and benzo[b]furan-2-boronic acid (1.2 g) weredissolved in DMF (15 ml) under a nitrogen flow, and the mixture wasadded with cesium carbonate (3.2 g) and tetrakistriphenylphosphinepalladium (0.29 g) and then allowed to react at 100° C. for 3 hours. Thereaction mixture was extracted with ethyl acetate and the extract wasconcentrated. The residue was recrystallized from ethyl acetate toobtain Compound VIII-8. Yield: 1.0 g (73%). H-NMR (CDCl₃) δ 8.95 (d,1H), 8.02 (d, 1H), 7.62 (d, 1H), 7.54 (d, 1H), 7.36-7.22 (m, 2H), 7.10(s, 1H), 2.40 (s, 3H), 1.38 (s, 6H)

Example 9 Syntheses of Compounds VII-9 and VII-10

When a reaction was performed by using 2-furanboronic acid or2-thiopheneboronic acid instead of the benzo[b]furan-2-boronic acid usedin the synthesis of Compound VII-8 under the same conditions as thosefor the synthesis of Compound VII-8, Compound VII-9 and Compound V-10were obtained. Structure of each was verified by mass spectrometry.

Example 10 Synthesis of Compound VII-12

Compound VII-11 was synthesized by following the route shown below.

(1) Synthesis of Compound VII-11a

A reaction was performed by using 4-methoxyphenylboronic acid instead ofthe benzo[b]furan-2-boronic acid used in the synthesis of Compound VII-8under the same conditions as those for the synthesis of Compound VII-8to obtained Compound VII-11a. H-NMR (DMSO-d₆) δ 8.59 (d, 1H), 8.08 (d,1H), 7.62 (d, 2H), 7.02 (d, 2H), 3.80 (s, 3H), 2.30 (s, 3H), 1.38 (s,6H)

(2) Synthesis of Compound VII-11b

Compound VII-11a (1.0 g) was dissolved in 47% aqueous hydrogen bromide(15 ml) and allowed to react at 130° C. for 2 hours. The reactionmixture was neutralized with 10% aqueous sodium hydroxide with icecooling and extracted with chloroform. The chloroform was evaporated,and the residue was recrystallized from isopropyl alcohol/hexane toobtain Compound VII-11b. Yield: 0.85 g (90%).

The structure was verified by mass spectrometry.

(3) Synthesis of Compound VII-11

Compound VII-11b (0.22 g) was dissolved in DMF (10 ml) and added withsodium hydride (35 mg) and propanesulfone (0.1 g) with ice cooling, andthen the mixture was allowed to react under ice cooling for 10 minutesand at room temperature for 20 minutes. The reaction mixture was addedwith isopropyl alcohol and a small amount of ethyl acetate to depositcrystals to obtain Compound VII-11. Yield: 0.30 g (87%).

The structure was verified by mass spectrometry.

Example 11 Synthesis of Compound VII-12

A reaction was performed by using p-tolylboronic acid instead of thebenzo[b]furan-2-boronic acid used in the synthesis of Compound VII-8under the same conditions as those for the synthesis of Compound VII-8to obtain Compound VII-12. H-NMR (DMSO-d₆) δ 8.59 (d, 1H), 8.12 (d, 1H),7.62 (d, 2H), 7.30 (d, 2H), 3.80 (s, 3H), 2.38 (s, 3H), 2.30 (s, 3H),1.35 (s, 6H).

Example 12 Synthesis of Compound VII-42

5-Chloro-2-hydrazinopyridine (35 g) and 3-methyl-2-butanone (100 ml)were mixed and allowed to react at 100° C. for 30 minutes. Afterevaporation of excess 3-methyl-2-butanone, the reaction mixture wasadded with 1,4-butanediol (100 ml) and allowed to react for 5 hours withheating at 240° C. The reaction mixture was cooled and subjected tosilica gel column chromatography without treatment. The solvent wasevaporated from the fraction containing the target substance, and thenthe residue was crystallized from hexane/ethyl acetate to obtainCompound VII-42 as light brown crystals. Yield: 14.0 g (29.5%). Meltingpoint: 118-120° C. H-NMR (CDCl₃) δ 8.50 (d, 1H), 7.70 (d, 1H), 2.40 (s,3H), 1.36 (s, 6H)

Example 13 Synthesis of Compound VII-46

2,6-Difluoropyridine (25 g), isopropyl alcohol (100 ml) and hydrazinehydrate (40 ml) were mixed and refluxed with heating for 3 hours. Theisopropyl alcohol was evaporated under reduced pressure, and the residuewas added with water. The deposited crystals were collected byfiltration, washed with water and dried to obtain6-fluoro-2-hydrazinopyridine (21.2 g, yield: 76.8%). Coloration wasobserved during drying, and accordingly, the product was used for thenext reaction immediately after the drying.

6-Fluoro-2-hydrazinopyridine (10 g) and 3-methyl-2-butanone (20 ml) weremixed and allowed to react at 80° C. for 15 minutes. After evaporationof excess 3-methyl-2-butanone, the reaction mixture was added withethylene glycol (20 ml) and allowed to react for 5 hours with heating at220° C. The reaction mixture was cooled and added with water and ethylacetate to extract the target substance in the organic layer. The ethylacetate was evaporated and the residue was subjected to silica gelcolumn chromatography. The solvent was evaporated from the fractioncontaining the target substance, and then the residue was crystallizedfrom hexane/ethyl acetate to obtain Compound VII-46. Yield: 3.2 g(22.8%). Melting point: 88-90° C.

Example 14 Synthesis of Compound VII-64

Compound VII-42 (4.5 g), DMF (40 ml), 3-hydroxy-1-propanethiol (4.0 g)and potassium t-butoxide (3.9 g) were mixed and refluxed with heatingunder a nitrogen flow for 5 hours. After completion of the reaction, DMFwas evaporated under reduced pressure, and the resulting brown oilysubstance was purified by silica gel column chromatography to obtain thetarget substance as a light yellow oily substance. Yield 1.0 g (17.9%)H-NMR (CDCl₃) δ 8.88 (d, 1H), 7.58 (d, 1H), 4.83 (b, 1H), 3.77 (t, 2H),3.05 (t, 2H), 3.36 (s, 3H), 1.90 (m, 2H), 1.36 (s, 6H)

Example 15 Synthesis of Compound VII-72

(1) Synthesis of 6-mercaptohexanoic acid ethyl ester

8-Bromohexanoic acid ethyl ester (250 g), thiourea (102.3 g), andisopropanol alcohol (500 ml) were mixed and refluxed with heating for 10hours. After cooling, isopropanol alcohol was evaporated under reducedpressure and the residue was added with hexane (500 ml) and stirred. Thehexane layer was removed by decantation, and the residue was added withpotassium carbonate (400 g), ethyl acetate (1 L), ethanol (1 L) andwater (2 L), and then the mixture was allowed to react at 75° C. under anitrogen flow for 6 hours. After completion of the reaction, thereaction mixture was added with ethyl acetate (1 L), and the layers wereseparated. The organic layer was concentrated and purified by silica gelcolumn chromatography. The target substance was obtained as a colorlessoily substance. Yield: 159 g (80.5%).

(2) Synthesis of Compound VII-72

Compound VII-42 (5 g) was dissolved in DMF (40 ml) and added with6-mercaptohexanoic acid ethyl ester (8 g). This solution was added withpotassium acetate (5.5 g) and refluxed with heating under a nitrogenflow for 8 hours. The reaction mixture was purified by silica gel columnchromatography to obtain Compound VII-72 as a colorless oily substance.Yield: 2.6 g (30.0%). H-NMR (CDCl₃) δ 8.57 (d, 1H), 8.40 (d, 1H), 4.12(q, 2H), 2.90 (m, 4H), 2.31 (s, 3H), 2.28 (m, 2H), 1.63 (m, 4H), 1.33(s, 6H), 1.25 (t, 3H)

A compound of the general formula (VI) can be synthesized by reacting acompound of the general formula (VII) with an alkylating agent. However,in the synthesis, alkylation of indolenine nitrogen may sometimes besimultaneously occurred together with the intended alkylation on thenitrogen of the pyridine. Purity of a target substance can be increasedby washing with hot isopropyl alcohol or ethyl acetate for most ofcompounds. Compounds for which washing is less effective may besubjected to the subsequent reaction as a mixture. When dyes are formed,crystallizability of target substances often increases, and in most ofdyes, impurities derived from indolenine nitrogen-alkylated compoundscan be removed by recrystallization. In the following examples, it wasdifficult to obtain compounds of the general formula (VI) in a highpurity, and therefore, structures of target substances were verified bymass spectrometry.

Example 16 Synthesis of Compound VI-1

Compound VII-1 (1 g) was dissolved in acetone (2 ml) and added withmethyl iodide (2 ml), and then the mixture was refluxed with heating for6 hours. The crystals deposited from the reaction mixture were washedwith isopropyl alcohol to obtain amorphous Compound VI-1. Yield: 0.64 g(70%) Mass (posi): m/e=355

Example 17 Synthesis of Compound VI-2

Compound VII-1 (1 g) was dissolved in acetone (2 ml) and added with2-methylpropanesulfone (2 ml), and then the mixture was refluxed withheating for 5 hours. The crystals deposited from the reaction mixturewere washed with acetone to obtain Compound VI-2. Yield: 0.71 g (65%)Mass (posi): m/e=476

Example 18 Synthesis of Compound VI-7

Compound VII-1 (1 g) was dissolved in acetone (2 ml) and added withethyl 6-bromohexanoate (2 ml), and then the mixture was refluxed withheating for 8 hours. When ethyl acetate was added to the reactionmixture, oily precipitates were formed. The supernatant ethyl acetatewas removed by decantation and the residue was washed several times withethyl acetate and dried under reduced pressure to obtain amorphousCompound VI-7. Yield: 0.70 g (76%) Mass (nega): m/e=483

Other compounds represented by the general formula (VI) weresuccessfully synthesized according to the synthesis methods of Examples16 and 18.

A compound represented by the formula (I) or the formula (II) can begenerally synthesized by the methods shown in the following schemes.

Scheme (A)

Method for Synthesizing a Symmetric Cyanine

Scheme (B)

Method for Synthesizing an Asymmetric and Merocyanine

Example 19 Synthesis of Compound I-1

Compound I-1 was synthesized according to the route of Scheme (B).Compound VI-1 (0.35 g), N,N′-diphenylformamidine (0.6 g) and aceticanhydride (5 ml) were mixed and allowed to react at 90° C. for 1 hour.The reaction mixture was added with ethyl acetate (50 ml), and thedeposited dye intermediate was separated. The resulting dye intermediateand Compound VI-7 (0.48 g) were dissolved in DMF (5 ml) and added withtriethylamine (0.5 ml) and acetic anhydride (0.2 ml), and the mixturewas allowed to react at room temperature for 2 hours. The reactionmixture was added with ethyl acetate to produce oily residue. After thesupernatant ethyl acetate was removed, the residue was purified bysilica gel chromatography to separate ethyl ester of Compound I-1.

The resulting ester compound was dissolved in 5% aqueous lithiumhydroxide and allowed to react at room temperature for 30 minutes toproduce the target Compound I-1. The reaction mixture was added withwater, chloroform, hydrochloric acid and tributylamine, and the layerswere evaporated. The chloroform layer containing the target substancewas washed twice with water and concentrated, and the residue was addedwith ethyl acetate to obtain tributylamine salt of Compound I-1. Thetributylamine salt was dissolved in methanol and added with potassiumacetate to obtain potassium salt of Compound I-1, and the resultingproduct was recrystallized from methanol to obtain Compound I-1 at anHPLC purity of 99% or higher. Yield: 120 mg (11%). Mass (nega): m/e=861Absorption maximum (methanol): 650 nm Molecular extinction coefficient:238000 Melting point: >300° C.

Example 20 Synthesis of Compound I-2

A reaction was performed under the same conditions as those in Example19 except that Compound I-2 was used instead of Compound I-1 used in thesynthesis of Compound I-1 to obtain Compound I-2. Mass (nega): m/e=1015Absorption maximum (methanol): 651 nm Molecular extinction coefficient:238000

Example 21 Synthesis of Compound I-3

A carboxylic acid derivative of Compound I-3 was synthesized under thesame conditions as those for the synthesis of Compound I-2. Theresulting carboxylic acid derivative was dissolved in a mixed solvent ofDMF and pyridine and added with N,N′-disuccinimdyl carbonate, and thenthe mixture was allowed to react at 40° C. for 2 hours. The reactionmixture was added with isopropyl alcohol and the resulting crystals werecollected by filtration. The crystals were purified by gel filtration(Sophadox G 10) to obtain Compound I-3. Mass (nega): m/e=1031 Absorptionmaximum (methanol): 651 nm Molecular extinction coefficient: 218000

Example 22 Synthesis of Compound I-11

Compound I-11 was synthesized by following the route shown below.

Compound I-11a was obtained by using Compound VI 0 and Compound VI-12according to the aforementioned method. Compound I-11a (45 mg) wasdissolved in DMF (4 ml) and added with potassium carbonate (0.2 g) andpropanesulfone (0.5 ml), and then the mixture was allowed to react at50° C. for 8 hours to attain sulfoalkylation. The reaction mixture wasadded with isopropyl alcohol and the resulting crystals were collectedby filtration. The crystals were dissolved in water and added withchloroform, tributylamine and hydrochloric acid and the layer wasseparated. The chloroform layer was concentrated under reduced pressure,and the resulting oily residue was added with 6% aqueous lithiumhydroxide and stirred at room temperature for 1 hour to performhydrolysis of the ester. Then, the reaction mixture was added withchloroform, tributylamine and hydrochloric acid an the layers wereseparated. The chloroform layer was concentrated and the resulting oilyreside was added with a methanol solution of potassium acetate to obtainCompound I-11 as crystals. Yield: 40 mg (69%) Mass (nega): m/e=1248Absorption maximum (methanol): 650 nm Molecular extinction coefficient:235000 Melting point: >300° C.

Example 23 Synthesis of Compound I-12

Compound I-12 was synthesized via the same synthetic route and under thesame conditions as those for Compound I-11 by using Compound VII-4 as astarting material. Mass (nega): m/e=1234 Absorption maximum (methanol):649 nm Molecular extinction coefficient: 233000 Melting point: >300° C.

Example 24 Synthesis of Compound I-13

Compound I-13 was synthesized by the synthetic route described below byusing Compound VII-4 and Compound VII-5 as starting materials.

The synthetic conditions for Compound I-13 were similar to those forCompound I-11. Mass (nega): m/e=1262 Absorption maximum (methanol): 649nm Molecular extinction coefficient: 233000 Melting point: >300° C.

Other dyes were also successfully synthesized according to the methodsof Examples 19 and 24.

Example 25 Comparison of Relative Fluorescence Quantum Yield

Relative fluorescence quantum yields of the compounds of the presentinvention are shown in Table 1. Measurement and calculation of relativequantum yield were performed according to the method described inJournal of Chemical Society, Faraday Trans., 92, 4917-4925 (1996). Thestructures of the conventional cyanine dye used as comparative dyes areshown below. As clearly shown by the results in Table 1, the dyes of thepresent invention are suitable for excitation by using an inexpensivehelium-neon laser light source (633 nm), and their fluorescenceintensity is two to three times stronger than that of the conventionaldyes.

TABLE 1 Relative Absorption Fluorescence fluorescence Compound No.maximum (nm) maximum (nm) quantum yield Comparative 641 660 0.21Compound 1 Comparative 645 664 0.27 Compound 2 I-1 649 669 0.68 I-2 649669 0.68 I-8 649 669 0.67 I-11 651 672 0.63 I-12 650 671 0.62 I-13 651672 0.63 I-14 649 671 0.63 I-15 650 671 0.62 I-16 635 652 0.65 I-17 634651 0.64 I-18 635 652 0.63 I-21 643 668 0.61 I-22 656 676 0.61 I-23 646662 0.63 I-24 633 651 0.64 I-25 630 547 0.63 I-26 650 667 0.59 I-27 635650 0.69 I-28 634 650 0.7 I-31 632 648 0.72 I-36 625 640 0.6 I-44 623635 0.58 I-50 640 660 0.58 I-54 638 662 0.64

Measurement method

(1) Concentration of ethanol solution of dye was adjusted so that thesolution had absorbance of 0.1 at 620 nm.

(2) Fluorescence spectrum was measured at an excellent wavelength of 620nm.

(3) Area value of fluorescence spectrum was calculated.

(4) Relative value was calculated based on the area value of CrystalViolet of which absolute fluorescence quantum yield was known (Φf=0.54).

Example 26 Evaluation of Aggregation Characteristic

Compounds of the present invention were dissolved in 50 mM Tris-HClbuffer (pH 8.0), 4×SSC (67 mM NaCl, 67 mM sodium citrate), 1 MChurch-phosphate buffer (0.5 M Na₂HOP₄/H₃PO₄, pH 7.2) and 1 M aqueoussodium chloride, and absorption spectrum patterns were evaluated. As aresult, it was revealed that all of the dyes described in specificexamples had a monomeric spectrum in an organic solvent as a mainabsorbance. In particular, Compounds I-1 to I-21 were absolutely freefrom aggregation which is observed for ordinary cyanine dyes.

Industrial Applicability

The compounds represented by the general formula (I), (II), (IV) and (V)of the present invention have properties of two to three-fold higherfluorescence intensity than conventional dyes and no aggregationtendency even in an aqueous medium at a high salt concentration, andthus they satisfy conditions as dyes for fluorescence-labeling.Therefore, they can be used as highly sensitive fluorescence-labelingagents for DNA sequencing or measurement of physiologically activesubstance or the like based on fluorescence immunoassay, or can be usedas fluorescence contract media and the like that are administered into ablood vessel to visualize information in vivo. Furthermore, thecompounds represented by the general formula (III), (IV) and (VII) areuseful as synthetic intermediates for preparation of the aforementionedcompounds.

What is claimed is:
 1. A compound represented by the following generalformula (IV):

wherein V¹, V², V³, V⁶, V⁷ and V⁸ represent a hydrogen atom or a groupselected from the group consisting of a halogen atom, an alkyl group, analkenyl group, an alkynyl group, an aryl group, a heterocyclic group,cyano group, hydroxy group, nitro group, carboxyl group, an alkoxygroup, an aryloxy group, a silyloxy group, a heterocyclyloxy group,, anacryloxy group, a carbamoyloxy group, an alkoxycarbonyloxy group, anaryloxycarbonyloxy group, an amino group (including an anilino group),an acylamino group, an aminocarbonylamino group, an alkoxycarbonylaminogroup, an aryloxycarbonyl-lamino group, a sulfamoylamino group, analkylsulfonylamino group, an arylsulfonylamino group, a mercapto group,an alkylthio group, an arylthio group, a heterocyclylthio group, asuifamoyl group, an alkylsulfinyl group, an arylsulfinyl group, analkylsulfonyl group, an arylsulfonyl group, an acyl group, anaryloxycaronyl group, an alkoycarbonyl group, a carbamoyl group, aphosphono group, a phosphonato group and a group that can form acovalent bond with a compound to be labeled (each of said group may besubstituted), provided that V¹, V² and V³ do not simultaneouslyrepresent a hydrogen atom, and provided that V¹ and V², V² and V³, V⁶and V⁷, and V⁷ and V⁸ may each independently form a saturated orunsaturated ring that may be substituted; R¹ and R²each independentlyrepresent a hydrogen atom or a group selected from the group consistingof an alkyl group, an aryl group and a heterocyclic group (each of saidgroup may be substituted); R³, R⁴, R⁵ and R⁶ each independentlyrepresent an alkyl group that may be substituted, and R³ and R⁴, and R⁵and R⁶ may each independently bind to each other to form a ring that maybe substituted; L¹, L² and L³ each independently represent a methinegroup that may be substituted; p represents 1, 2 or 3; M represents acounter ion; and q represents a number required to neutralize a chargeof the molecule, wherein at least one of V¹, V² and V³ is an aryl groupsubstituted with a sulfo group or a salt thereof, a heterocyclic groupsubstituted with a sulfo group or a salt thereof, or an alkynyl groupsubstituted with a sulfo group or salt thereof.